A comparative analysis of 23 structures of the amyloidogenic protein transthyretin

Self-assembly of the human plasma protein transthyretin (TTR) into unbranched insoluble amyloid fibrils occurs as a result of point mutations that destabilize the molecule, leading to conformational changes. The tertiary structure of native soluble TTR and many of its disease-causing mutants have been determined. Several independent studies by X-ray crystallography have suggested structural differences between TTR variants which are claimed to be of significance for amyloid formation. As these changes are minor and not consistent between the studies, we have compared all TTR structures available at the protein data bank including three wild-types, three non-amyloidogenic mutants, seven amyloidogenic mutants and nine complexes. The reference for this study is a new 1.5 A resolution structure of human wild-type TTR refined to an R-factor/R-free of 18.6 %/21.6 %. The present findings are discussed in the light of the previous structural studies of TTR variants, and show the reported structural differences to be non-significant.     

Mechanisms of transthyretin cardiomyocyte toxicity inhibition by resveratrol analogs

The transthyretin amyloidoses are a subset of protein misfolding diseases characterized by the extracellular deposition of aggregates derived from the plasma homotetrameric protein transthyretin (TTR) in peripheral nerves and the heart. We have established a robust disease-relevant human cardiac tissue culture system to explore the cytotoxic effects of amyloidogenic TTR variants. We have employed this cardiac amyloidosis tissue culture model to screen 23 resveratrol analogs as inhibitors of amyloidogenic TTR-induced cytotoxicity and to investigate their mechanisms of protection. Resveratrol and its analogs kinetically stabilize the native tetramer preventing the formation of cytotoxic species. In addition, we demonstrate that resveratrol can accelerate the formation of soluble non-toxic aggregates and that the resveratrol analogs tested can bring together monomeric TTR subunits to form non-toxic native tetrameric TTR.     

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe --> Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.     

Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity

Amyloid formation of the plasma protein transthyretin (TTR) has been linked to familial amyloid polyneuropathy and senile systemic amyloidosis. Binding of ligands within its natural hormone binding site can stabilize the tetrameric structure and impair amyloid formation. We have recently shown that the flavonoid luteolin stabilizes TTR in human plasma with a very high selectivity. Luteolin, however, is inactivated in vivo via glucuronidation for which the preferred site is the hydroxy group at position 7 on its aromatic A-ring. We have evaluated the properties of two luteolin variants in which the 7-hydroxy group has been exchanged for a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut). Using an in vitro model, based on human liver microsomes, we verified that these modifications increase the persistence of the drug. Crystal structure determinations show that 7-Cl-Lut binds similarly to luteolin. The larger MeO substituent cannot be accommodated within the same space as the chlorine or hydroxy group and as a result 7-MeO-Lut binds in the opposite direction with the methoxy group in position 7 facing the solvent. Both 7-Cl-Lut and 7-MeO-Lut qualify as high-affinity binders, but in contrast to luteolin, they display a highly non-specific binding to other plasma components. The binding of the two conformations and the key-interactions to TTR are discussed in detail. Taken together, these results show a proof-of-concept that the persistence of luteolin towards enzymatic modification can be increased. We reveal two alternative high-affinity binding modes of luteolin to TTR and that modification in position 7 is restricted only to small substituents if the original orientation of luteolin should be preserved. In addition, the present work provides a general and convenient method to evaluate the efficacy of TTR-stabilizing drugs under conditions similar to an in vivo environment.     

Production of recombinant human transthyretin with biological activities toward the understanding of the molecular basis of familial amyloidotic polyneuropathy (FAP).

Transthyretin (TTR) is a plasma protein interacting with thyroxine T4 and retinol binding protein (RBP). Several variants of TTR with single amino acid substitutions have been identified as the major components of the amyloid fibrils of familial amyloidotic polyneuropathy (FAP), a fetal, autosomal dominant genetic disease. The elucidation of the molecular nature of the variants distinct from that of the wild-type TTR is crucial for understanding the amyloidogenesis in FAP, but our understanding is very poor mainly because of the unavailability of pure variant TTRs. In the present study, we used an Escherichia coli OmpA secretion vector (Ghrayeb et al., 1984) and achieved an effective production of the variant TTRs related to FAP including Met-30, Ile-33, Ala-60, Tyr-77, Met-111, and Ile-122 types. The variant TTRs produced in this system were efficiently secreted to the culture media. The chemical analysis showed that the secreted TTR (Met-30 type) has the same N-terminus as the native one. IEF analyses also indicated that the secreted product is properly processed as assessed by its pI. Furthermore, the secreted TTR was shown to have biological activities, namely, the thyroxin binding activity and the ability to associate with retinol binding protein, indicating that the secreted TTR polypeptide is properly folded. The present work also demonstrated that the processing/secretion of the recombinant TTR molecules in E. coli was strongly affected by single amino acid substitutions.     

Detection and identification of protein variants and adducts in blood and tissues: an application of soft ionization mass spectrometry to clinical diagnosis

The detection and identification of protein variants and abnormally increased modified proteins are important for clinical diagnosis. We applied soft ionization mass spectrometry (MS) to analyze proteins in blood and tissues from various patients. Over the past 8 years, we diagnosed 132 cases (55 kinds) of variant proteins including hemoglobin (Hb), transthyretin (TTR), and Cu/Zn-superoxide dismutase (SOD-1), using MS as the leading technology. Of these variants, eight were new, and nine were the first cases in Japan. Some abnormal Hb cause diseases, and most of them cause erroneous levels of glycated Hb, HbA1c, i.e., a popular index of diabetes. Most of the variant TTR causes amyloidotic polyneuropathy. Variant SOD-1 causes amyotrophic lateral sclerosis. We first showed that immunoprecipitation by a specific antiserum is a reliable and simple method to prepare protein from sera and tissues for analysis by matrix-assisted laser desorption time-of-flight MS, and liquid chromatography-electrospray ionization MS (LC-ESI-MS). The use of this technology has become widespread. Using an immunoprecipitated target protein and LC-ESI-MS, we showed that the ratios of tetra-, di- and a-sialo-transferrin from two cases of congenital glycoprotein deficient syndrome were clearly distinguishable from those of control samples. We first reported a unique modified form of TTR, that is, S-sulfonated TTR, which increased markedly and specifically in three cases with molibdenum cofactor deficiency. We proposed that S-sulfonated TTR is a useful marker for screening this disease. ESI-MS was successfully used for the accurate determination of HbA1c, and we clarified the extent of discrepancies between the HbA1c value measured by conventional methods and the accurate values for samples containing various Hb variants determined by the MS method.     

From molecular variant to disease: initial steps in evaluating the association of transthyretin M119 with disease.

Traditionally, clinical research has sought to determine the molecular basis of clinical signs and symptoms. Increasingly, the traditional process will be reversed, as many structural protein variants are elucidated as a result of powerful PCR-based methods. Herein we describe a variant of transthyretin (TTR) found by direct genomic sequencing and illustrate the utility of PASA (PCR amplification of specific alleles) in the initial characterization of such variants. TTR is an intriguing protein of unknown function, but deposition of mutant TTR produces familial amyloidotic polyneuropathy (FAP). We identify a carrier of a variant TTR in which threonine119 is changed to methionine (T119----M). T119 is invariant in five mammalian species, suggesting that this residue is important for normal protein function. To determine the frequency of the M119 variant, individuals of northern- and western-European descent were rapidly screened by generating a PASA assay for the sequence change. Four additional individuals were found to be heterozygous for the mutation, for a total of five M119 alleles in 1,666 genes (1/333). Clinical records, initial clinical interviews, and family history of these patients hint at a high frequency of early-onset venous insufficiency and perhaps mild renal dysfunction. Haplotype analysis on the heterozygotes could be performed, despite the absence of samples from relatives, by performing "double PASA." The haplotype data suggest that the M119 variant derives from a common ancestor. The putative functional deficiency caused by TTR M119 should be most marked in the homozygotes, who can be calculated to occur in 1/100,000 conceptions. If viable, these individuals may provide important clues about the physiological role of TTR. Although the nature (if any) of disease caused by TTR M119 remains to be defined, the genetic and clinical data indicate that this mutation does not cause FAP. Future family studies can determine whether the heterozygous state for TTR M119 cosegregates with a disease or trait.     

Hepatic production of transthyretin L12P leads to intracellular lysosomal aggregates in a new somatic transgenic mouse model

Transthyretin (TTR) is a plasma and cerebrospinal fluid (CSF)-circulating homotetrameric protein. More than 100 point mutations have been identified in the TTR gene and several are related with amyloid diseases. Here we focused our attention in the TTR L12P variant associated with severe peripheral neuropathy and leptomeningeal amyloidosis. By using different cell lines derived from tissues specialized on TTR synthesis, such as the hepatocyte and the choroid plexus expressing WT, V30M, or L12P TTR variants we analyzed secretion, intracellular aggregation and degradation patterns. Also, we used liver-specific AAV gene transfer to assess expression of the L12P variant in vivo. We found the following: (i) decreased secretion with intracellular aggregation of TTR L12P in hepatoma cells relative to WT and V30M variant; this differential property of TTR L12P variant was also observed in mice injected with L12P AAV vector; (ii) differential N-glycosylation pattern of L12P variant in hepatoma cell lysates, conditioned media and mouse sera, which might represent an escape mechanism from ERAD degradation; (iii) intracellular L12P TTR aggregates mainly localized to lysosomes in cultured cells and liver; and (iv) none of the above findings were present in choroid plexus derived cells, suggesting particular secretion/quality control mechanisms that might contribute to leptomeningeal amyloidosis associated with the L12P variant. These observations open new avenues for the treatment of TTR associated leptomeningeal amyloidosis.     

Oxidation inhibits amyloid fibril formation of transthyretin

The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on protein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing protein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril formation, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by comparing the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxidative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril formation for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by approximately 76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures.     

Biochemical basis for retinol deficiency induced by the I41N and G75D mutations in human plasma retinol-binding protein

Retinol-binding protein (RBP) is the retinol-specific carrier protein present in plasma, where it circulates almost entirely bound to thyroxine-binding transthyretin (TTR). Recently, depressed plasma retinol and RBP levels in carriers of the I41N and G75D RBP point mutations have been reported. We show here that although recombinant human N41 and D75 RBPs can form complexes with retinol and TTR in vitro, the retinol-mutated RBP complexes are significantly less stable than human normal holo-RBP, as revealed by the markedly facilitated retinol release by mutated holo-RBPs to phospholipid membranes, in accordance with the location of mutated residues inside the RBP retinol-binding cavity. Taken together, the data are consistent with the I41N and G75D point mutations being the cause of an altered interaction of retinol with RBP, resulting in a remarkably reduced stability of the retinol-RBP complex, which in turn can lead to the lowering of plasma retinol and RBP levels.     

Continuous spectrometric assays for glutaminyl cyclase activity

A high-throughput mass spectrometric immunoassay system for the analysis of proteins directly from plasma is reported. A 96-well format robotic workstation was used to prepare antibody-derivatized affinity pipette tips for subsequent use in the extraction of specific proteins from plasma and deposition onto 96-well format matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) targets. Samples from multiple individuals were screened with regard to the plasma protein transthyretin (TTR), followed by analysis of the same plasma samples for the transthyretin-associated transport protein, retinol-binding protein (RBP). Analyses were able to detect the presence of posttranslationally modified TTR and RBP, as well as a mutation present in the TTR of one individual. Subsequent analyses of wild-type and mutated TTR using enzymatically active MALDI-TOF MS targets were able to identify the site and nature of the point mutation. The approach represents a rapid (approximately 100 samples/2 h, reagent preparation-to-data) and accurate means of characterizing specific proteins present in large numbers of individuals for proteomic and clinical/diagnostic purposes.     

D18G transthyretin is monomeric,aggregation prone,and not detectable in plasma and cerebrospinal fluid: a prescription for central nervous system amyloidosis?

Over 70 transthyretin (TTR) mutations facilitate amyloidosis in tissues other than the central nervous system (CNS). In contrast, the D18G TTR mutation in individuals of Hungarian descent leads to CNS amyloidosis. D18G forms inclusion bodies in Escherichia coli, unlike the other disease-associated TTR variants overexpressed to date. Denaturation and reconstitution of D18G from inclusion bodies afford a folded monomer that is destabilized by 3.1 kcal/mol relative to an engineered monomeric version of WT TTR. Since TTR tetramer dissociation is typically rate limiting for amyloid formation, the monomeric nature of D18G renders its amyloid formation rate 1000-fold faster than WT. It is perplexing that D18G does not lead to severe early onset systemic amyloidosis, given that it is the most destabilized TTR variant characterized to date, more so than variants exhibiting onset in the second decade. Instead, CNS impairment is observed in the fifth decade as the sole pathological manifestation; however, benign systemic deposition is also observed. Analysis of heterozygote D18G patient's serum and cerebrospinal fluid (CSF) detects only WT TTR, indicating that D18G is either rapidly degraded postsecretion or degraded within the cell prior to secretion, consistent with its inability to form hybrid tetramers with WT TTR. The nondetectable levels of D18G TTR in human plasma explain the absence of an early onset systemic disease. CNS disease may result owing to the sensitivity of the CNS to lower levels of D18G aggregate. Alternatively, or in addition, we speculate that a fraction of D18G made by the choroid plexus can be transiently tetramerized by the locally high thyroxine (T(4)) concentration, chaperoning it out into the CSF where it undergoes dissociation and amyloidogenesis due to the low T(4) CSF concentration. Selected small molecule tetramer stabilizers can transform D18G from a monomeric aggregation-prone state to a nonamyloidogenic tetramer, which may prove to be a useful therapeutic strategy against TTR-associated CNS amyloidosis.     

Trapping the monomer of a non-amyloidogenic variant of transthyretin: exploring its possible use as a therapeutic strategy against transthyretin amyloidogenic diseases

Transthyretin (TTR) is a 127-residue homotetrameric beta-sheet-rich protein that transports thyroxine in the blood and cerebrospinal fluid. The deposition of fibrils and amorphous aggregates of TTR in patients' tissues is a hallmark of TTR amyloid disease. Familial amyloidotic polyneuropathy is a hereditary form of TTR amyloidosis that is associated with one among 80 different variants of TTR. The most aggressive variants of TTR are V30M, L55P, and A25T, and the propensity to undergo aggregation seems to be linked to tetramer stability. T119M is a very stable, non-amyloidogenic variant of TTR. Here we show that the combination of high hydrostatic pressure with subdenaturing concentrations of urea (4 m) at 1 degrees C irreversibly dissociates T119M into monomers in less than 30 min in a concentration-dependent fashion. After pressure and urea removal, long lived monomers are the only species present in solution. We took advantage of the slow reassociation kinetics of these monomers into tetramers to produce heterotetramers by mixing the T119M monomers with the tetramers of the aggressive mutants of TTR. Our data show that T119M monomers can be successfully incorporated into all of these tetramers even when the exchange is performed in a more physiological environment such as human plasma; these monomers render the resultant heterotetramers less amyloidogenic. The data presented here are relevant for the understanding of T119M folding and association reactions and provide a protocol for producing T119M monomers that function as inhibitors of TTR aggregation when incorporated in to tetramers. This protocol may provide a new strategy for treating TTR diseases for which there is no therapy available other than liver transplantation.     

Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

TTR (transthyretin) amyloidoses are diseases characterized by the aggregation and extracellular deposition of the normally soluble plasma protein TTR. Ex vivo and tissue culture studies suggest that tissue damage precedes TTR fibril deposition, indicating that early events in the amyloidogenic cascade have an impact on disease development. We used a human cardiomyocyte tissue culture model system to define these events. We previously described that the amyloidogenic V122I TTR variant is cytotoxic to human cardiac cells, whereas the naturally occurring, stable and non-amyloidogenic T119M TTR variant is not. We show that most of the V122I TTR interacting with the cells is extracellular and this interaction is mediated by a membrane protein(s). In contrast, most of the non-amyloidogenic T119M TTR associated with the cells is intracellular where it undergoes lysosomal degradation. The TTR internalization process is highly dependent on membrane cholesterol content. Using a fluorescent labelled V122I TTR variant that has the same aggregation and cytotoxic potential as the native V122I TTR, we determined that its association with human cardiomyocytes is saturable with a K_D near 650 nM. Only amyloidogenic V122I TTR compete with fluorescent V122I for cell-binding sites. Finally, incubation of the human cardiomyocytes with V122I TTR but not with T119M TTR, generates superoxide species and activates caspase 3/7. In summary, our results show that the interaction of the amyloidogenic V122I TTR is distinct from that of a non-amyloidogenic TTR variant and is characterized by its retention at the cell membrane, where it initiates the cytotoxic cascade.     

Cys-10 mixed disulfide modifications exacerbate transthyretin familial variant amyloidogenicity: a likely explanation for variable clinical expression of amyloidosis and the lack of pathology in C10S/V30M transgenic mice?

The marked variation in clinical expression and age of familial amyloid disease onset is not well understood. One possibility is that metabolite modification(s) of a disease-associated mutant protein can change the energetics and propensity for misfolding, influencing the disease course. Each subunit of the transthyretin (TTR) tetramer has a single Cys residue that can exist in the SH form or as a mixed disulfide with the amino acid Cys or the peptide glutathione or fragments of the latter. The stability and amyloidogenicity of the clinically most important TTR variants (V30M and V122I) in their SH oxidation state were compared with those of their mixed disulfide adducts. All the Cys-10 mixed disulfide conjugates exhibited substantially decreased protein stability (urea, pH 7) and a higher rate and extent of amyloidogenesis (slightly acidic conditions). We also investigated the amyloidogenicity and stability of a C10S/V30M TTR double mutant which lacks the ability to make mixed disulfides, but retains the disease-associated V30M mutation. Unlike V30M TTR, this double mutant is nonamyloidogenic in transgenic mice. Our in vitro data reveal that the C10S/V30M and V30M TTR homotetramers have identical amyloidogenicity and stability, implying that Cys-10 mixed disulfide formation enhances amyloidogenesis in V30M transgenic mice. Given the high proportion of TTR subunits having mixed disulfide modifications in human plasma ( approximately 50%), and the data within demonstrating their increased amyloidogenicity, we submit that disulfide metabolite modifications have the potential to influence the course of amyloidoses, including TTR amyloidoses caused by mutations.     

Simultaneous characterization of SNPs and N-glycans from multiple glycosylation sites of intact β-2-glycoprotein-1 (B2GP1) by ESI-qTOF-MS

The highly glycosylated ß-2-glycoprotein-1 (B2GP1), also called apolipoprotein H, is a 50 kDa human plasma protein with four or five N-glycosylation sites. Glycosylation of B2GP1 can impact auto antibody recognition leading to the development of antiphospholipid syndrome (APS), which can result in miscarriages or thrombosis. Next to its glycosylation different genetic variants are known to increase the risk of suffering from APS. Here we show that ESI-q/TOF-MS of intact B2GP1 can be used to analyze genetic variants and glycosylation simultaneously. After enrichment of B2GP1 from 16 different plasma samples and subsequent ESI-MS measurement of the intact protein, we detected five different SNPs in our samples either homozygous or heterozygous. The dominant glycan composition shows four biantennary, fully sialylated glycan structures, with a relative proportion of about 30%. We also detected compositions with one or two triantennary glycan structures in lower amounts and fucosylated species with one or two fucosyl residues. Two of our samples showed an unreported partially occupied fifth glycosylation site presumably arising from the presence of SNP variant S88N. Our method allows a fast determination of genetic variants and glycan compositions of human B2GP1 to be potentially used as diagnostic marker.     

Delineation of in vivo assembled multiprotein complexes via biomolecular interaction analysis mass spectrometry

Biomolecular interaction analysis mass spectrometry (BIA-MS) is a multiplexed bioanalytical approach used in analysis of proteins from complex biological mixtures. It utilizes surface-immobilized ligands for protein affinity retrieval, surface plasmon resonance for monitoring the ligand-protein interaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry for revealing the masses of the biomolecules retrieved by the ligand. In order to explore the utility of BIA-MS in delineation of multiprotein complexes, an in vivo assembled protein complex comprised of retinol binding protein (RBP) and transthyretin (TTR) was investigated. Antibodies to RBP and TTR were utilized as ligands in the analysis of the protein complex present in human plasma. The RBP-TTR complex was retrieved by the anti-RBP antibody as indicated by the presence of both RBP and TTR signals in the mass spectra. RBP signals were not observed in the mass spectra of the material retained on the anti-TTR derivatized surface. In addition, the mass-specific detection in BIA-MS allowed detection of RBP and TTR analyte variants.     

Presence of N‐glycosylated transthyretin in plasma of V30M carriers in familial amyloidotic polyneuropathy: an escape from ERAD

Familial amyloid polyneuropathy (FAP) is an autosomal dominant disease characterized by deposition of amyloid related to the presence of mutations in the transthyretin (TTR) gene. TTR is mainly synthesized in liver, choroid plexuses of brain and pancreas and secreted to plasma and cerebrospinal fluid (CSF). Although it possesses a sequon for N‐glycosylation N‐D‐S at position 98, it is not secreted as a glycoprotein. The most common FAP‐associated mutation is TTR V30M. In a screening for monoclonal antibodies developed against an amyloidogenic TTR form, we detected a distinct TTR with slower electrophoretic mobility in Western of plasma from carriers of the V30M mutation, not present in normal plasma. Mass spectrometry analyses of this slower migrating TTR (SMT) identified both wild‐type and mutant V30M; SMT was undetectable upon N‐glycosidase F treatment. Furthermore, SMT readily disappeared in the plasma of V30M ‐ FAP patients after liver transplantation and appeared in plasma of transplanted domino individuals that received a V30M liver. SMT was also detected in plasma, but not in CSF of transgenic mice for the human V30M mutation. A hepatoma cell line transduced to express human V30M did not present the SMT modification in secretion media. Glycosylated TTR was absent in fibrils extracted from human kidney V30M autopsy tissue or in TTR aggregates extracted from the intestine of human TTR transgenic mice. Studies on the metabolism of this novel, glycosylated TTR secreted from FAP liver are warranted to provide new mechanisms in protein quality control and etiopathogenesis of the disease.     

X-ray absorption spectroscopy reveals a substantial increase of sulfur oxidation in transthyretin (TTR) upon fibrillization

Transthyretin (TTR) amyloid fibrils are the main component of the amyloid deposits occurring in Familial Amyloidotic Polyneuropathy patients. This is 1 of 20 human proteins leading to protein aggregation disorders such as Alzheimer's and Creutzfeldt-Jakob diseases. The structural details concerning the association of the protein molecules are essential for a better understanding of the disease and consequently the design of new strategies for diagnosis and therapeutics. Disulfide bonds are frequently considered essential for the stability of protein aggregates and since in the TTR monomers there is one cysteine residue, it is important to determine unambiguously the redox state of sulfur present in the fibrils. In this work we used x-ray spectroscopy to further characterize TTR amyloid fibrils. The sulfur K-edge absorption spectra for the wild type and some amyloidogenic TTR variants in the soluble and fibrillar forms were analyzed. Whereas in the soluble proteins the thiol group from cysteine (R-SH) and the thioether group from methionine (R-S-CH(3)) are the most abundant forms, in the TTR fibrils there is a significant oxidation of sulfur to the sulfonate form in the cysteine residue and a partial oxidation of sulfur to sulfoxide in the methionine residues. Further interpretation of the data reveals that there are no disulfide bridges in the fibrillar samples and suggest conformational changes in the TTR molecule, namely in strand A and/or in its vicinity, upon fibril formation.     

Human plasma retinol-binding protein (RBP4) is also a fatty acid-binding protein

RBP4 (plasma retinol-binding protein) is the 21?kDa transporter of all-trans retinol that circulates in plasma as a moderately tight 1:1 molar complex of the vitamin with the protein. RBP4 is primarily synthesized in the liver but is also produced by adipose tissue and circulates bound to a larger protein, transthyretin, TTR, that serves to increase its molecular mass and thus avoid its elimination by glomerular filtration.This paper reports the high resolution three-dimensional structures of human RBP4 naturally lacking bound retinol purified from plasma, urine and amniotic fluid. In all these crystals we found a fatty acid molecule bound in the hydrophobic ligand-binding site, a result confirmed by mass spectrometry measurements.In addition we also report the 1.5?Å resolution structures of human holo-RBP4 and of the protein saturated with palmitic and lauric acid and discuss the interaction of the fatty acids and retinol with the protein.